Genetic engineering of plants through transgenic approaches has rapidly become a diverse and promising field. Transformation techniques are applied both to study plant development and physiology, and in attempts to generate plants with advantageous agricultural properties.
For example, much effort has focused on improving crop production by transforming plants with herbicide or pesticide resistance genes. When grown under field conditions, the resulting transgenic plants can tolerate and survive applications of herbicides or pesticides which would destroy non-transformed plants. Alternatively, plants themselves have been genetically engineered to express products which are shown to act as natural insecticides such as Bacillus thuringensis toxin proteins (see U.S. Pat. No. 5,380,831, issued Jan. 10, 1995).
Genetic engineering has also been attempted to generate plants with altered compositions of primary metabolites such as starch, sugar, and oils. For example, a bacterial levan sucrase gene was transformed into plants to increase levels of fructans in the resulting transformant. In another approach, the activity of certain enzymes involved in the metabolic synthesis of oils has been decreased using antisense technology in order to design vegetable oils exhibiting the desired properties. (Reviewed in Knauf, 1995, Curr. Opin. Biotech. 6: 165-70).
In addition to attempts to alter primary metabolic compositions of plants (i.e. carbohydrates, oils, amino acids and proteins), it would also be desirable to alter secondary metabolite compositions. Secondary metabolites are those specialized plant products required by cells in small amounts (e.g. hormones), or are highly specialized biomolecules (e.g. nucleotides, pigments, toxins, antibiotics, and alkaloids). Generally, secondary metabolites can be defined as compounds that have no recognized role in the maintenance of fundamental life processes of the cell, but which may perform other advantageous functions for the organism as a whole. (See for example, Bell, 1981, "The Physiological Role(s) of Secondary (Natural) Products" in The Biochemistry of Plants, v. 7, p. 1, Academic Press, Inc.)
The production of useful secondary metabolites from plants and plant cell cultures is an important aspect of plant technology. About 25% of the prescription drugs used in the industrialized world contain ingredients extracted from higher plants (Tyler, V. E., 1988, Planta Med. 54: 95-100). The most prominent group of plant substances used in medicine are the alkaloids (Nickell, L. G., 1980, "Products," Plant tissue culture as a source of biochemicals, Staba, E. J., Ed., Boca Raton, CRC Press Inc., 235-269). Alkaloids are secondary metabolites which protect plants against phytophagocytosis by higher organisms or invasion by pathogens (Robins, R. J. et al., 1991, Planta Med. 57: 27-35). Plant alkaloids are generally derived from simple amino acids that interact with acetate and terpenoid units and undergo aromatic hydroxylations (Robins, supra). Examples of plant alkaloids are nicotine, scopolamine, hyoscyamine, ajmalicine, serpentine, piperine, leucenol, mimosine, ricinine, pelletierine, cocaine, hygrine, lupinine and anagyrine.
Several examples of introducing a new metabolic pathway or redirecting previously existing ones to increase production of secondary metabolites have been described (Bailey, supra; Berlin, J. et al., 1994, Stud. Plant Sci. 4: 57-81; Lilius, G., Holmberg, N. and Bulow, L., 1996, Bio/Technology 14: 177-180). However, apparently all of these genetic engineering approaches entail either the up-regulation of a limiting enzyme in the pathway (through transformation and expression of the cloned gene), or the down-regulation of competing enzymatic pathways (through anti-sense technology). (See for review, Robins et al., 1991, Planta. Med. 57: 27-35.)
Plant cell suspension cultures have often been used for industrial production of plant secondary metabolites. During in vitro culture, a critical parameter influencing secondary metabolite formation, especially in scale-up cultures, is the level of dissolved oxygen. For instance, it has been demonstrated that a high dissolved oxygen concentration promotes the production of ajmalicine in Catharantus roseus batch cultures (Schaltmann, J. E. et al., 1995, Biotech. Bioeng. 45: 435-439). Similarly, increased alkaloid levels with increasing aeration have been observed in Berberis wilsonae (Breuling, M. et al., 1985, Plant Cell Rep. 4: 220-223). From these results it has been suggested that oxygen is a limiting substrate in plant tissue culture secondary metabolism, as well as for primary metabolism, especially for industrial scale-up processes. However, this need for increased oxygen has always been thought to be a need for extracellular oxygen during fermentation batch processes.
Aerobic metabolism has been successfully enhanced in fermentation microorganisms and cultured mammalian cells by engineering the host to express the Vitreoscilla hemoglobin gene (VHb) (Khosla, C., Bailey, J. E., 1988, Nature 331: 633-635; Khosla, C., Bailey, J. E., 1988, Mol. Gen. Genet. 214: 158-161; DeModena, supra; Chen, W. et al., 1994, Biotechnol. Prog. 10: 308-313; Pendse, G. J. and Bailey, J. E., 1994, Biotechnol. Bioeng. 44: 1367-1370). This metabolic engineering strategy has been shown effective, for example, in increasing total cell protein synthesis by oxygen-limited Escherichia coli (Khosla, C. et al., 1990, Bio/Technology 8: 849-853 and U.S. Pat. No. 5,049,493, issued Sep. 17, 1991), improving lysine yield and titer in cultivations of Corynebacterium glutamicum (Sander, F. et al., 1994, Proc. 6th Eur. Congress Biotechnol., Alberghina, L., Frontali, L. and Sensi, P. (eds.) Elsevier Science B. V., Amsterdam 607-610), and in increasing actinorhodin and cephalosporin C production in Streptomyces coelicolor and Acremonium chrysogenum, respectively (Magnolo, S. K. et al., 1991, Bio/Technology 9: 473-476; DeModena, J. A. et al., 1993, Bio/Technology 11: 926-929).
Hemoglobin and globin-like proteins exist ubiquitously in mammals, and are less frequently found in plants. The few plant hemoglobin-like molecules described, which include lupin and soybean leghemoglobin, are largely thought to be associated with nitrogen-fixation activities of these plants, although some researchers claim that hemoglobin-like proteins occur in the roots of all plants. However, in no case have these proteins been overexpressed in plants. Although several major plant metabolic pathways are known to be oxygen-dependent, including chlorophyll and heme biosynthesis, fatty acid desaturation and cysteine, glycine and serine biosynthesis, oxygen has not been thought to be a limiting factor in metabolism in intact plants. Rather, the observed effects of dissolved oxygen on metabolism in plant tissue cultures has been attributed to mass transport problems during large-scale, or even batch culture, fermentations (see Schlatmann et al., 1991, supra.). Thus, until the present invention, the advantages of altering useable levels of intracellular oxygen in intact plants have not been recognized.